Treatment of diabetes

ABSTRACT

The present invention provides methods for using a killed malaria parasite or an extract thereof in the treatment of non-insulin dependent diabetes mellitus (NIDDM).

The present invention relates to treatment of diabetes mellitus,specifically treatment of non-insulin dependent diabetes mellitus.

Diabetes is sub-divided on clinical grounds into insulin-dependent andnon-insulin dependent diabetes mellitus (IDDM and NIDDM respectively,also known as Type 1 and Type 2 respectively). The two forms of thedisease are distinguished by a number of features.

In IDDM there is profound insulin deficiency such that even the lowlevels of insulin which would normally prevent lipolysis and cytogenesiscannot be sustained. Without replacement of insulin, IDDM patientsbecome ketotic and die. IDDM patients therefore generally show highlevels of glucose and low levels of insulin. As IDDM progresses, thepancreatic islets are damaged or destroyed, and less and less insulincan be produced.

NIDDM is a common and complex disorder which results from a combinationof defects in insulin secretion and impaired insulin sensitivity inperipheral tissues. NIDDM is characterized by hyperglycaemia in both thefasted and fed states, variable degrees of hyperinsulinaemia andobesity. Current therapy includes diet, sulphonylurea to enhance insulinsecretion, insulin itself, and biguanides to reduce insulin resistance.There is a need for new antidiabetic agents, since biguanides are quitetoxic while sulphonylurea is ineffective in patients with severelyimpaired islet cell function, and after 10 years of treatment, 50% ofpatients will have become resistant.

Clinically, the two forms of diabetes are often viewed as two differentdiseases, and entirely separate treatments are needed for the two forms.In general treatments for the two forms of the disease do not overlap.

In humans, the normal level of glucose in the blood is about 7 mmol/l.Patients are said to be hypoglycaemic at levels of less than about 2.2mmol/l and hyperglycaemic at levels of above about 10-11 mmol/l.

One of the complications of malaria infection is hypoglycaemia, althoughthe mechanism responsible has not been firmly established. In patientstreated with quinine, the drop in glucose levels is often attributed tothe hyperinsulinaemic action of quinine, for example by Phillips et alin Q.J. Med. 1993 volume 86, pp 233-240. A few cases in whichhypoglycaemia and hyperinsulinaemia preceded treatment with quinine havebeen reported, for example by Looareesuwna et al in Lancet 1985 ii:4-8.

A single case of a diabetic patient who contracted malaria is reportedby Shalev et al in Postgrad. Med. J. 1992 vol. 68, pp 281-282. Thepatient still became hypoglycaemic during the course of the malariainfection.

Infection of normal mice with blood-stage P. yoelii and P. chabaudimalaria can induce hypoglycaemia in the normal mice (Elased et al, Clin.Exp. Immunol. 1995, vol. 99, pp 440-444). It is thought that this effectmay be due to induction of a burst of insulin which reduces the glucoselevels. In the same article, it was reported that malaria infection ininsulin-dependent diabetes mellitus (IDDM) induced by administration ofstreptozotocin induced a profound drop in blood glucose and restoredinsulin secretion, although severely diabetic mice (again induced bystreptozotocin) remained hyperglycaemic with no changes in insulinlevels. This supports the theory that the effect is due to induction ofa burst of insulin, since mice severely affected with IDDM will have fewor no residual islets which can be stimulated to produce insulin.

In NIDDM, the insulin levels are already raised, and there issignificant insulin resistance. The inventors have unexpectedly foundthat administration of malaria parasites to mice which provide a modelof human NIDDM will result in lowering of glucose levels. This isentirely unexpected, since previously it was thought that the reductionof glucose levels seen on giving malaria parasites to Type 1 (IDDM)diabetic mice was due to the induction of a burst of insulin. Themechanism of action in NIDDM treatment will inevitably be different.

A further complication of NIDDM is obesity. It has long been known thatincreasing body weight is associated with increasing levels of insulinresistance, seen in NIDDM. The interaction between obesity and diabetesis explored by Sigal et al in Current Opinion in Endocrinology andDiabetes 1996, volume 3, pp. 3-9. Control of NIDDM can often be achievedby regulation of the diet, and of total food intake. However, this isoften unsuccessful as individuals may find it difficult to maintain adiet and to cut food intake.

We have found that administration of malaria parasites or an extractthereof to a mouse model of human NIDDM results in reduced voluntaryfood intake, with consequent weight loss.

Although not wishing to be bound by this theory, it appears possiblethat the malaria parasite or extract thereof may act synergisticallywith the high levels of insulin in the NIDDM sufferer to enhance glucosetransport since when normal mice are treated with the killed parasite,the reduction in glucose levels was much less prolonged.

The invention provides the use of killed malaria parasites or an extractthereof in the preparation of a medicament for the treatment ofnon-insulin dependent diabetes mellitus (NIDDM). The malaria parasitemay be any organism responsible for malaria infection. Examples includePlasmodium yoelii, P. falciparum, P. vinckei, P. vivax, P. chabaudi, P.berghei, P. knowlesi and P. coatenyi. The killed malaria parasites orextracts thereof are particularly effective when collected at the bloodstage of infection.

The medicament may be provided in the form of killed malaria parasitesor an extract of the parasites, for example fixed in formalin. Othersuitable forms include soluble malaria preparations prepared by lysis ofparasitised red cells by detergents such as Triton X100 or N-octylglucoside, which may be followed by fractionation, for example onmolecular weight or charge-based columns, and also preparations derivedfrom supernatants of overnight culture of parasitised blood.

The preparation of parasites or the extract can be administered in avariety of dosage forms, for example orally such as in the form oftablets, capsules, sugar- or film-coated tablets, liquid solutions orsuspensions or parenterally, for example intramuscularly, intravenouslyor subcutaneously. The parasites or extracts thereof may therefore begiven by injection or infusion.

The dosage of the parasite preparation or the extract depends on avariety of factors including the age, weight and condition of thepatient and the route of administration.

It is envisaged that the malaria parasites or active ingredients derivedtherefrom will be given in a dosage of from 10⁷ to 10¹¹, preferably 10⁸to 10¹⁰ parasites or parasite equivalents. By “parasite equivalents”herein is meant the number of parasites needed to prepare an extract.

The killed malaria parasites or the extract may be given to the patientin a single dose, which it is expected will be sufficient to reducehyperglycaemia for a period of up to 24 hours. Alternatively, lowerdosages may be provided a number of times per day, for example 3 or 4times daily. It is further envisaged that a single, larger dose could beprovided for longer term treatment.

The parasites or extracts thereof are formulated for use aspharmaceutical or veterinary compositions also comprising apharmaceutically or veterinarily acceptable carrier or diluent. Thecompositions are typically prepared following conventional methods andare administered in a pharmaceutically or veterinarily suitable form.

For example, the solid oral forms may contain, together with the activematerial, diluents such as lactose, dextrose, saccharose, cellulose,corn starch or potato starch; lubricants such as silica, talc, stearicacid, magnesium or calcium stearate and/or polyethylene glycols; bindingagents such as starches, arabic gums, gelatin, methylcellulose,carboxymethylcellulose, or polyvinyl pyrrolidone; disintegrating agentssuch as starch, alginic acid, alginates or sodium starch glycolate;effervescing mixtures; dye-stuffs; sweeteners; wetting agents such aslecithin, polysorbates, laurylsulphates. Such preparations may bemanufactured in known manner, for example by means of mixing,granulating, tabletting, sugar coating, or film-coating processes.

Liquid dispersions for oral administration may be syrups, emulsions andsuspensions. The syrups may contain as carrier, for example, saccharoseor saccharose with glycerol and/or mannitol and/or sorbitol. Inparticular a syrup for diabetic patients can contain as carriers onlyproducts, for example sorbitol, which do not metabolise to glucose orwhich only metabolise a very small amount to glucose. The suspensionsand the emulsions may contain as carrier, for example a natural gum,agar, sodium alginate, pectin, methylcellulose, carboxymethylcelluloseor polyvinyl alcohol.

Suspensions or solutions for intramuscular injections may contain,together with the active compound, a pharmaceutically acceptable carriersuch as sterile water, olive oil, ethyl oleate, glycols such aspropylene glycol, and, if desired, a suitable amount of lidocainehydrochloride. Solutions for intravenous injection or infusion maycontain a carrier, for example, sterile water which is generally Waterfor Injection. Preferably, however, they may take the form of a sterile,aqueous, isotonic saline solution. Alternatively, a material may beencapsulated within liposomes.

The invention also relates to a method of treatment of NIDDM whichcomprises administering to a patient in need thereof a preparationcomprising the killed malaria parasites or an extract thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the effect of formalin fixed P. yoelli on the blood glucoseof obese diabetic mice (aged 10-12 weeks).

FIG. 2 shows the effect of malaria lysate on insulin secretion fromisolated rat pancreatic islets.

FIGS. 3 and 4 shows the effect of parasite on the food intake andglucose levels respectively, of db/db mice.

The invention is further illustrated by the following examples.

EXAMPLES

Parasites

Infections with the lethal YM line of Plasmodium yoelii (FFYM; from Dr.A. Holder, National Institute of Medical Research, London, England) wereinitiated by i.v. injection of 10⁴ parasitized erythrocytes into normalmice and parasitaemia was determined from blood films stained withGiemsa. In the case of P. berghei ANKA (PB; from Dr. N. Wedderburn) 10⁶parasitized rbc were injected intraperitoneally. Plasmodium falciparum(PF) was obtained from Dr. M. Blackman (National Institute of MedicalResearch, London, England).

Parasitised blood (>90% parasitaemia) was extracted and washed threetimes in PBS, and the pellet was lysed in 0.01% saponin PBS and washedthree times, fixed with 0.06% formalin overnight and washed a furtherthree times before use. This formalin fixed malaria parasite preparation(FFMP) was administered i.v. in saline.

Blood with a parasitaemia of at least 90% was extracted and washed,lysed in 0.01% saponin, and extracted for three hours in 0.5% TritonX100 or 0.7% N-octyl glucoside and then microfuged. The lysate was runon a Sephacryl S.200 column (Pharmacia) and fractions within the firstpeak (mol. wt. 200-170 kDa) were pooled as Peak 1. Parasite preparationswere either injected via the tail vein or fed orally via gastric tube.

Mice

Genetically obese diabetic mice (C57 Bl/Ks db/db) were obtained fromHarlan Olac Ltd. Bicester, UK. These mice are a recognised animal modelof human NIDDM. Properties of the mice are reviewed by E. Shafrir inDiabetes and Metabolism (Paris) 1996, vol. 22, pp 122-131. The db/dbmice are spontaneously hyperphagic insulin oversecretors even beforeweaning and then become obese and hyperinsulinaemic within the firstmonth of age. Hyperglycaemia develops somewhat later, becoming severe atthree to four months. The db/db mice exhibit marked insulin resistance.

The experiments described below were carried out on mice aged 8 to 12weeks, when both blood glucose and insulin levels are markedly raised(glucose 20.4 mmol/l, insulin 54.9 ng/ml). Normal heterozygous (db/+)litter mates were used as controls (glucose 6.6 mmol/l, insulin 2.8ng/ml). For some comparisons, normal (C57Bl×Balb/c) F1 mice bred in theanimal colony of our laboratory were used.

Measurement of Blood Glucose

Glucose concentrations were determined from a drop of tail bloodcollected between 10 am and midday, using Glucostix and an AmesGlucometer (Miles Ltd., Stbke Poges, England) according to themanufacturer's instructions. Results in mmol/l are expressed asmeans±SE.

Determination of Immunoreactive Insulin

Blood was collected from the trunk following decapitation, inheparinized tubes. Plasma was separated by centrifugation and frozen at−20° C. Immunoreactive insulin (IRI) concentrations were determined in50 μl volumes in duplicate by the double-antibody radioimmunoassaytechnique (kit supplied by ICN Biomedicals, Irvine, Calif.) and acrystalline rat insulin standard (Novo Research Institute, Bagsvaerd,Denmark). Plasma insulin in ng/ml is expressed as mean±SE.

Isolation and Incubation of Pancreatic Islets

Islets were isolated from the pancreas of male Sprague-Dawley rats(250-300 g) maintained on a standard light-dark cycle, by a modificationof the method of Lacy & Kostianovsky (1967) using collagenase. Two ratswere used per assay. Islets were preincubated in batches of 5-6 for 30min with Krebs bicarbonate buffer (1.0 ml. pH 7.3 to 7.4, 95% O₂, CO₂,37° C.) containing 3 mmol/l glucose. The preincubation medium wasremoved and then replaced with 1.0 ml of Krebs bicarbonate with orwithout malaria lysate and incubated for a further 60 min. A sample ofincubate (200 μ) was removed and stored at −20° C. until assay forinsulin.

Statistics

Comparisons were made by Student's t test. Values of P<0.01 wereconsidered significant.

Experiment 1

A single injection of 5×107 formalin fixed P. yoelii (FFYM) parasiteswas administered to db/db obese mice, a model of type 2, non-insulindependent diabetes mellitus. A group of nine diabetic (db/db) mice wereinjected and another group of six left uninjected (“controls”). Bloodglucose was measured at 2, 4, 6, 8, 24, and 30 hours as shown in FIG. 1.

It can be seen that the single injection of FFYM lowered the bloodglucose from a level or 17 to 18 mmol/l to a normal value of about 7mmol/l. This normoglycaemia persisted for a period of 24 hours.

Experiment 2

A detergent lysate of P. yoelii was prepared as described above, andPeak 1 obtained by S.200 fractionation. Addition of this fraction to ratpancreatic islets as described above in the presence of glucose, asdescribed above, stimulated the release of insulin. Results arepresented in FIG. 2.

Experiment 3

Formalin fixed P. yoelli (FFYM) parasites (10⁸ in 0.2 ml) were givenorally to a group of 6 db/db mice, and 0.2 ml of phosphate bufferedsaline (PBS) to another group. Blood glucose was measured as describedabove, and the average quantity of food eaten per day was recorded. Themice given the malaria parasites showed a drop in food intake and asignificant decrease of blood glucose. Results are shown in FIGS. 3 and4.

What is claimed is:
 1. A method of treatment of NIDDM comprisingadministering to a patient in need thereof, a preparation comprising akilled malaria parasite or a lysate thereof or a fraction of saidlysate.
 2. A method according to claim 1, wherein the malaria parasiteis selected from the group consisting of Plasmodium yoelii, P.falciparum, P. vinckei, P. vivax, P. chabaudi, P. berghei, P. knowlesiand P. coatneyi.
 3. A method according to claim 1 wherein the malariaparasite is collected at the blood stage of infection.
 4. A methodaccording to claim 1 wherein the preparation provides a dosage of from10⁷ to 10¹¹ parasites or parasite equivalents.
 5. A method according toclaim 2 wherein the malaria parasite is collected at the blood stage ofinfection.
 6. A method according to claim 2 wherein the preparationprovides a dosage of from 10⁷ to 10¹¹ parasites or parasite equivalents.7. A method according to claim 3 wherein the preparation provides adosage of from 10⁷ to 10¹¹ parasites or parasite equivalents.
 8. Amethod according to claim 2, wherein the malaria parasite comprisesPlasmodium yoeli.
 9. A method according to claim 2, wherein the malariaparasite comprises P. falciparum.
 10. A method according to claim 2,wherein the malaria parasite comprises P. vinckei.
 11. A methodaccording to claim 2, wherein the malaria parasite comprises P. vivax.12. A method according to claim 2, wherein the malaria parasitecomprises P. chabaudi.
 13. A method according to claim 2, wherein themalaria parasite comprises P. berghei.
 14. A method according to claim2, wherein the malaria parasite comprises P. knowlesi.
 15. A methodaccording to claim 2, wherein the malaria parasite comprises P.coatneyi.